Transcriptomes of the major human pancreatic cell types.

AIMS/HYPOTHESIS: We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. METHODS: Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations. RESULTS: Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function. CONCLUSIONS/INTERPRETATION: This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

Characterization and retroviral transduction of an early human lymphomyeloid precursor assayed in nonswitched long-term culture on murine stroma.

In the hierarchy of human hematopoietic progenitors, long-term culture-initiating cells (LTC-IC) and extended LTC-IC belong to the earliest cell populations that can be assayed in vitro. We report the identification of a multipotential lymphomyeloid progenitor detected in a nonswitch culture system. We observed the emergence of CD33+ myeloid and CD19+ B-lymphoid cells following plating of lineage-depleted (Lin-) CD34 -enriched or purified CD34+ CD38- cord blood cells on MS-5 stroma in the absence of exogenous cytokines. Both CD19+ CD20- pro-B and CD19+ CD20+ pre-B lymphocytes coexist with myeloid cells in long-term culture. A limiting dilution approach was used to show that a single CD34+ CD38- cell can generate lymphomyeloid progeny in conventional (5-week) and extended (10-week) cultures. Most of the clones in long-term culture or extended long-term culture contained not only lymphoid and myeloid cells, but also myeloid clonogenic progenitors. A high proportion of CD34+ CD38- cells gave rise to lymphomyeloid clones after 5 and 10 weeks of culturing (up to 48% and 16%, respectively), which distinguishes the assay reported here from those using switch culture conditions. We performed retroviral gene transfer experiments involving 1-3 days of exposure of Lin CD34+ -enriched cells to virus encoding enhanced green fluorescent protein. Monitoring of gene transfer efficiency into LTC-IC by enhanced green fluorescent protein fluorescence showed that it is possible to achieve marking of lymphomyeloid LTC-IC, albeit to a lesser extent than myeloid-restricted LTC-IC.

Speciation, conductivities, diffusivities, and electrochemical reduction as a function of water content in mixtures of hydrated chromium chloride/choline chloride.

We report experiments and simulations to understand the factors that control chromium (Cr(3+)) electrodeposition from ionic liquid solutions. Speciation, conductivities and diffusivities in mixtures of trivalent chromium chloride, water and choline chloride (CrCl3/xH2O/yChCl) were computed from molecular dynamics simulations and compared to measured ultraviolet-visible spectra, conductivities from electrical impedance spectroscopy, and cyclic voltammograms. Computed changes in Cr(3+) first solvation shell and conductivity with solution composition qualitatively agree with experimental observations. The Cr(3+) first solvation shell contains predominantly H2O and Cl(-) and the proportion of the two ligands changes with the relative bulk concentrations of each. Conductivities and diffusivities are observed to be functions of these composition variables. Variations in observed reduction current are primarily determined by dynamical properties and are less influenced by speciation.

In vivo dynamics of human stem cell repopulation in NOD/SCID mice.

Primitive human hematopoietic cells can be assayed on the basis of their ability to repopulate immune-deficient NOD/SCID mice and have been termed SCID repopulating cells (SRCs). The in vivo biological fate of individual SRCs can be tracked by following the unique retroviral insertion site in the progency of transduced SRCs. Distinct human SRCs were identified that differ in the proliferative and self-renewal capacity indicating that the primitive cell compartment is functionally heterogeneous.

Enhancement of oxyanion and diatrizoate reduction kinetics using selected azo dyes on Pd-based catalysts.

Azo dyes are widespread pollutants and potential cocontaminants for nitrate; we evaluated their effect on catalytic reduction of a suite of oxyanions, diatrizoate, and N-nitrosodimethylamine (NDMA). The azo dye methyl orange significantly enhanced (less than or equal to a factor of 5.24) the catalytic reduction kinetics of nitrate, nitrite, bromate, perchlorate, chlorate, and diatrizoate with several different Pd-based catalysts; NDMA reduction was not enhanced. Nitrate was selected as a probe contaminant, and a variety of azo dyes (methyl orange, methyl red, fast yellow AB, metanil yellow, acid orange 7, congo red, eriochrome black T, acid red 27, acid yellow 11, and acid yellow 17) were evaluated for their ability to enhance reduction. Hydrogenation energies of azo dyes were calculated using density functional theory and a volcano relationship between hydrogenation energies and reduction rate enhancement was observed. A kinetic model based on Brønsted-Evans-Polanyi (BEP) theory matched the volcano relationship and suggests sorbed azo dyes enhance reduction kinetics through hydrogen atom shuttling between reduced azo dyes (i.e., hydrazo dyes) and oxyanions or diatrizoate. This is the first research that has identified this synergetic effect, and it has implications for designing more efficient catalysts and reducing Pd costs in water treatment systems.

Distinct classes of human stem cells that differ in proliferative and self-renewal potential.

The composition of the human hematopoietic stem cell compartment is poorly understood due to the absence of experimental tools with which to characterize the developmental program of individual stem cells. We report here that human stem cells differ markedly in their repopulation capacity and self-renewal potential, as determined using nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice transplanted with retrovirally transduced cord blood stem cells, called SCID-repopulating cells (SRCs). Clonal stem cell analysis based on the identification of unique retroviral integration sites within serial bone marrow aspirates showed that repopulation was generally oligoclonal with extensive variability in the lifespan and proliferative capacity of individual SRCs. Most clones contributed to human cell engraftment for several weeks after transplantation and then disappeared but others appeared later and persisted. Further evidence for stem cell heterogeneity was found in the secondary transplantation capacity of SRCs. These data point to the existence of different classes of human stem cells with variable self-renewal potential and short- or long-term repopulating capacity.

Expansion of human cord blood CD34(+)CD38(-) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function.

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin(-) CD34(+)CD38(-) cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin(-)CD34(+) CD38(-) cells and SRC, CD34(+)-enriched lineage-depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34(+)CD38(-) cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP(+) human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34(+)CD38(-) phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34(+)CD38(-) phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34(+)CD38(+) cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells.

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.

Transduction of human CD34+ CD38- bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors.

The major limitations of Moloney murine leukemia virus (MoMLV)-based vectors for human stem cell applications, particularly those requiring bone marrow (BM) stem cells, include their requirement for mitosis and retroviral receptor expression. New vectors based upon lentiviruses such as HIV-1 exhibit properties that may circumvent these problems. We report that novel third-generation, self-inactivating lentiviral vectors, expressing enhanced green fluorescent protein (EGFP) and pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G), can efficiently transduce primitive human repopulating cells derived from human BM and cord blood (CB) tested by the SCID-repopulating cell (SRC) assay. Highly purified CD34+ CD38- CB or BM cells were efficiently transduced (4-69%) and stably expressed in EGFP for 40 days in culture following infection for only 24 h without fibronectin, polybrene, or cytokines. Nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice transplanted with transduced cells from either CB or BM donors were well engrafted, demonstrating maintenance of SRC during the infection procedure. Serially obtained femoral BM samples indicated that the proportion of EGFP+ cells within both myeloid and lymphoid lineages was maintained or even increased over time, averaging 42.3 +/- 6.6% for BM donors and 23.3 +/- 7.2% for CB at 12 weeks. Thus, the third-generation lentivectors readily transduce human CB and BM stem cells, under minimal conditions of ex vivo culture, where MoMLV-based vectors are ineffective. Since CB is inappropriate for most therapeutic applications, the efficient maintenance and transduction of BM-derived SRC during the short infection procedure are notable advantages of lentivectors.